Methods of making a molecular detection chip having a metal oxide silicon field effect transistor on sidewalls of a micro-fluid channel

ABSTRACT

A molecular detection chip including a metal oxide silicon-field effect transistor (MOSFET) on sidewalls of a micro-fluid channel and a molecular detection device including the molecular detection chip are provided. A molecular detection method, particularly, qualification methods for the immobilization of molecular probes and the binding of a target sample to the molecular probes, using the molecular detection device, and a nucleic acid mutation assay device and method are also provided. The formation of the MOSFET on the sidewalls of the micro-fluid channel makes easier to highly integrate a molecular detection chip. In addition, immobilization of probes directly on the surface of a gate electrode ensures the molecular detection chip to check for the immobilization of probes and coupling of a target molecule to the probes in situ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/239,736,filed Sep. 25, 2002 now U.S. Pat. No. 7,235,389, which is a 35 U.S.C.§371 national stage application of International PCT Application No.PCT/KR02/00746 filed Apr. 23, 2002, which claims priority from KoreanApplication No. 2001-21752, filed Apr. 23, 2001, Korean Application No.2001-29729 filed May 29, 2001 and Korean Application No. 2001-78010filed Dec. 11, 2001. The disclosure and content of each of theseapplications are incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a molecular detection chip including ametal oxide semiconductor field effect transistor (MOSFET) formed onsidewalls of a micro-fluid channel, and a molecular detection deviceemploying the molecular detection chip. Also, the present inventionrelates to a molecular detection method using the molecular detectiondevice, and more particularly, to a quantitative detection method forthe immobilization of molecular probes or the binding of molecularprobes and a target sample. The present invention relates to a nucleicacid mutation assay device constructed by incorporating a thermalcontrol and detection unit including a heater and a thermal sensor intothe molecular detection device, and a nucleic acid mutation assay methodusing the nucleic acid mutation assay device.

2. Description of the Related Art

The disclosure of the human DNA sequence by the completion of genomeproject has accelerated researches into the function of diverse nucleicacids and proteins coded by the nucleic acids, and at the same time,increased the need for biosensors for easy detection of biomoleculessuch as nucleic acids and proteins.

Biosensors capable of sensing biomolecules using electrical signals aredisclosed in U.S. Pat. Nos. 4,238,757, 4,777,019, 5,431,883, and5,827,482. U.S. Pat. No. 4,238,757 discloses a field effect transistor(FET) designed to have a source and drain, including a layer ofantibodies specific to a particular antigen. The concentration ofantigens in a sample solution is measured from drain current variationsover time using the FET.

U.S. Pat. No. 4,777,019 discloses a FET in which a gate is formed acrossdoped source and drain regions, and a nucleotide complementary to atarget nucleotide to be measured is bound to the top of the gate.

U.S. Pat. No. 5,431,883 discloses a FET in which a thin film ofphthalocyanin, an organic insulating material capable of being changedto be conductive through reactions with chemical species, is formed toconnect a gate and a drain.

U.S. Pat. No. 5,827,482 discloses a biosensor including two FETsconnected in parallel, each having respective gates to which molecularreceptors sensitive to different materials are bound for improvedsensitivities.

However, currently available biosensors are all formed as conventionalplanar surface FETs each having a source, a drain, and a channel layeron the surface of a planar substrate so that high integration of thebiosensors is restricted. In addition, it is difficult to selectivelyimmobilize biomolecular probes on a limited region. Accordingly,immobilization of probes on the FETs is performed by using a separatefabrication apparatus in the fabrication of the FETs. However, theresulting probes are weakly immobilized on the FETs so that binding to atarget molecule cannot be detected with high sensitivity. In addition, aconsiderable time is required to check for the probe immobilization,thereby increasing the overall time consumption for target moleculedetection.

Therefore, there is an increasing need for a new biosensor which is easyto highly integrate, ensures stable immobilization of probes and in-situconfirmation of the probe immobilization, and can detect binding of atarget molecule with high sensitivity.

Biochips refer to chips having highly immobilized biomolecular probes,such as DNA, proteins, etc., to be analyzed on substrates and are usedfor the analysis of a gene expression profile, genetic defects, aprotein profile, and reaction patterns. Biochips can be categorized intoa microarray chip having immobilized probes on a solid substrate and alab-on-a-chip having immobilized probes on a micro-channel according tothe type of immobilization of the probes, and into a DNA chip, a proteinchip, etc., according to the kinds of probes.

Most DNA chips currently available are manufactured based on a spottingor photolithography technique. A DNA chip is manufactured byimmobilizing only a single DNA strand that can react with a target DNA,as a probe on a particular substrate using chemical bonds, and detectsthe target DNA from the reaction. In manufacturing such a DNA chip,immobilization of probe DNAs greatly affects the reliability andreproducibility of products, and thus it needs to be accuratelycontrolled. However, techniques in current use fail to accuratelyquantify the immobilized biomolecules.

Conventional spotting chips or photolithography chips can adjust thequantity of probes to a certain level on a volume basis in themanufacturing process, but have poor accuracy and reproducibility foruse as commercial products for the diagnosis of diseases. In particular,for the investigation of particular DNA expression, more accurateimmobilization of probe DNAs is required. Despite the need for anaccurate immobilization technique, one has not been established yet dueto technical problems in manufacturing processes and cost concerns.

To overcome the conventional problems, the present inventors haveconducted research and completed the present invention where the voltageand current characteristics of a DNA chip were measured using a MOSFETsensor in the DNA chip so that immobilization and hybridization of probeDNAs could be accurately detected. As a result, a DNA chip capable ofmeasuring the immobilization and hybridization of probe DNAs at the sametime can be manufactured for commercial uses without an increase in themanufacturing cost.

Single nucleotide polymorphism (SNP) of nucleic acids, which is a singlebase pair variation of human DNA between individuals, is the most commonDNA sequence polymorphism (about 1 per 1000 bases). SNP affects theimmune system of individuals and thus can be effective for diagnosing,treating, and preventing inherited diseases. Therefore, there is a needfor a rapid and convenient detection method of SNP originating fromindividual or racial genetic differences and immunities.

Common SNP detection methods are based on temperature-dependentseparation of double-stranded DNA. Double-stranded DNA is separated intotwo single strands at a temperature greater than about 95° C. Based uponthese characteristics, the sequence of mutated DNA can be identified.However, this method needs discrete systems and apparatuses for eachstep and cannot be applied for real-time DNA separation and immediatedetection through accurate and precise temperature control.

U.S. Pat. No. 6,171,850 entitled “Integrated Devices and Systems forperforming Temperature Controlled Reactions and Analyses” discloses useof a heat exchanger in controlling the internal and externaltemperatures of individual reactors. The reaction system includes aheater and at least one heat exchanger. This reaction system is merelyfocused on temperature control of a plurality of reactors. Also, theinclusion of the heat exchanger is not advantageous in DNA detection andimprovement of fluid channel characteristics.

U.S. Pat. No. 6,174,676 entitled “Electrical Current for ControllingFluid Parameters in Micro-channels” discloses a variety of heaters and afluid channel equipped with an electrically controlled heater andcooler. This patent is restricted to the temperature control apparatusfor PCR without description of a device for detecting temperature-basedseparation of DNA duplex.

U.S. Pat. No. 5,683,657 entitled “DNA meltometer” discloses a nucleicacid analytical device including a temperature control chamber forcarrying a buffer solution while being kept at a predeterminedtemperature, a heating and cooling unit for controlling the temperatureof the temperature control chamber, and a labeling and detecting unitfor labeling thermally denaturated double-stranded DNA at a temperatureT_(m) with fluorescent materials and detecting them. In the DNAmeltometer, the DNA detecting unit is separated from the temperaturecontrol chamber, thereby complicating the overall system and causing adelay in the detection step.

To address these problems, the present inventors have developed a DNAmutation (SNP) assay device which ensures DNA detection as well astemperature adjustment in a variety of ways based upon the fact that adouble-stranded DNA is separated into two single strands at an increasedtemperature. According to the present invention, whether DNA isseparated or not can be identified in real time using a DNA detectionunit disposed on a fluid channel.

SUMMARY OF THE INVENTION

It is a first object of the present invention to provide a moleculardetection chip that is easy to highly integrate and can detect theimmobilization of probes and binding of a target molecule in situ withina short period of time.

It is a second object of the present invention to provide a method forfabricating the molecular detection chip.

It is a third object of the present invention to provide a moleculardetection device employing the molecular detection chip.

It is a fourth object of the present invention to provide a quantitativedetection method of the immobilization of molecular probes using themolecular detection device.

It is a fifth object of the present invention to provide a quantitativedetection method of the binding of molecular probes and a target sampleusing the molecular detection device.

It is a sixth object of the present invention to provide a new nucleicacid mutation assay device in which at least one thermal control anddetection unit is formed in a micro-fluid channel by microfabrication sothat DNA mutations can be detected in real time through accurate andprecise temperature control.

It is a seventh object of the present invention to provide an effectivenucleic acid mutation (single nucleotide polymorphism; SNP) assay methodusing the nucleic acid mutation assay device.

To achieve the first object of the present invention, there is provideda molecular detection chip comprising: a semiconductor substrate; amicro-fluid channel serving as a flow path of a molecular sample andformed in the semiconductor substrate, and a metal oxide siliconfield-effect transistor (MOSFET) on sidewalls of the micro-fluidchannel.

In the present invention, the term “sidewalls” of the micro-fluidchannel is intended to include convex corners and bottoms as well as theactual sidewalls of the micro-fluid channel.

It is preferable that a gate electrode of the MOSFET is formed of a thingold (Au) layer. It is preferable that thiol-substituted probes areimmobilized as self-assembled monolayers on the surface of the gateelectrode.

To achieve the second object of the present invention, there is provideda method for fabricating a molecular detection chip, the methodcomprising forming an oxide layer on the surface of a semiconductorsubstrate. Next, a micro-fluid channel having at least one convex corneris formed by etching the surface of the semiconductor substrate, and thesidewalls of the micro-fluid channel are doped with impurity ions toform an impurity diffusion region. A channel region of a MOSFET isdefined by etching a portion of the impurity diffusion region formed onthe sidewalls of the micro-fluid channel using an etching solution. Anoxide layer is formed on the channel region, and a gate electrode isformed on the channel region using gold (Au), thereby resulting in amolecular detection chip according to the present invention.

It is preferable that defining the channel region is performed byselectively etching the impurity diffusion region along the at least oneconvex corner. In defining the channel region, it is preferable that anetch stop point is determined by measuring current level variations withapplication of reverse bias voltage to the semiconductor substrate.

To achieve the third object of the present invention, there is provideda molecular detection device comprising: a substrate; a molecular-sampleloading unit formed on the surface of the substrate; at least onemicro-fluid channel having one end connected to the molecular-sampleloading unit and serving as a flow path for a molecular sample; and aMOSFET sensor connected to the other end of the micro-fluid channel andon which a molecular probe is immobilized.

Preferably, the MOSFET sensor is implemented with a molecular detectionchip according to the present invention. In this case, it is preferablethat the molecular detection chip is manufactured separately from amolecular detection kit including the substrate, the molecular sampleloading unit, and the micro-fluid channel, and then mounted on a chipmount region located at the end of the micro-fluid channel of themolecular detection kit.

The present invention also provides a molecular sample detection methodcomprising providing molecular probes through a molecular-sample loadingunit formed on the surface of the substrate of a molecular detectiondevice. Next, the molecular probes are immobilized on the surface of agate electrode of a MOSFET formed on sidewalls of a micro-fluid channelof the molecular detection device, the micro-fluid channel having oneend connected to the molecular-sample loading unit and the other endconnected to the MOSFET so that the molecular probes provided throughthe sample loading unit move through the micro-fluid channel into theMOSFET. Following this, current-voltage characteristics of the gateelectrode are measured. After providing a target molecular samplethrough the molecular-sample loading unit, the target molecular sampleis reacted with the molecular probes while moving through the fluidchannel of the molecular detection device. Finally, current-voltagecharacteristics of the gate electrode are measured to detect the targetmolecular sample based on the change in the current-voltagecharacteristics with respect to the result measured before loading thetarget molecular sample.

It is preferable that the molecular detection method further comprisesremoving the molecular probes that are not immobilized on the gateelectrode by loading a cleaning solution into the molecular-sampleloading unit after immobilizing the molecular probes on the surface ofthe gate electrode, and removing the target sample that is not reactedwith the molecular probes by loading a cleaning solution into themolecular-sample loading unit after reacting the target molecular samplewith the molecular probes.

In the present invention, the molecular probes or the target molecularsample may comprise nucleic acids, proteins, enzyme substrates,adjuvants, and oligosaccharides. Preferably, the nucleic acids comprisesingle-stranded deoxyribonucleic acids (DNAs), single-strandedribonucleic acids (RNAs), and single-stranded peptide nucleic acids(PNAs). Preferably, the proteins comprise agonists to cell membranereceptors, antagonists to cell membrane receptors, toxins, virusepitopes, hormones, peptides, enzymes, and monoclonal antibodies. Morepreferably, the molecular probes are single-stranded nucleic acids, andthe target molecular sample is single-stranded nucleic acids capable ofbeing hybridized with the molecular probes.

The molecular detection chip and device according to the presetinvention are advantageously easy to highly integrate and can detectimmobilization of probes and coupling of a target molecule to the probesin situ.

To achieve the fourth object of the present invention, there is provideda quantification method of the immobilization of molecular probes, themethod comprising: providing the molecular probes into themolecular-sample loading unit of the molecular detection devicedescribed above; immobilizing the molecular probes on the surface of agate electrode of the MOSFET sensor by allowing the molecular probes tomove along the micro-fluid channel; and measuring current-voltagecharacteristics of the gate electrode.

To achieve the fifth object of the present invention, there is provideda quantification method of the binding of molecular probes and a targetmolecular sample, the method comprising: (a) providing the molecularprobes into the molecular-sample loading unit of the molecular detectiondevice described above; (b) immobilizing the molecular probes on thesurface of a gate electrode of the MOSFET sensor by allowing themolecular probes to move along the micro-fluid channel; (c) measuringcurrent-voltage characteristics of the gate electrode; (d) providing thetarget molecular sample into the molecular-sample loading unit; (e)binding the target molecular sample to the molecular probes immobilizedon the surface of the gate electrode of the MOSFET sensor by allowingthe target molecular sample to move along the micro-fluid channel; and(f) measuring current-voltage characteristics of the gate electrode andcomparing the result of the measurement with the current-voltagecharacteristics measured in step (c).

According to the present invention, by measuring the current-voltagecharacteristics of the gate electrode of a MOSFET sensor installed inthe molecular detection device, immobilization of probes andhybridization of a target sample to the immobilized probes can bequantitatively measured. For a DNA chip, immobilization of probe nucleicacids, such as single-stranded deoxyribonucleic acids (DNAs),single-stranded ribonucleic acids (RNAs), and single-stranded peptidenucleic acids (PNAs), and hybridization of target nucleic acids to theprobe nucleic acids can be quantitatively measured.

To achieve the sixth object of the present invention, a heater, athermal sensor, and a DNA sensor formed as a MOSFET sensor are built asan assembly in a micro-fluid channel for temperature-based DNAseparation and detection. According to the present invention, thetemperature, which affects DNA structure, is adjusted using the heaterand the thermal sensor, and at the same time, whether the DNA is single-or double-stranded can be detected in real time.

In particular, the present invention provides a nucleic acid mutationassay device comprising: a substrate; a sample loading unit formed onthe surface of the substrate; a micro-fluid channel directly connectedto the sample loading unit to serve as a sample flow path; and at leastone thermal control and detection unit formed in the micro-fluid channeland including a MOSFET sensor for nucleic acid immobilization, a heater,and a thermal sensor.

It is preferable that the heater and the thermal sensor are locatedadjacent to the MOSFET sensor and control a temperature which issignificant to the nucleic acid immobilized on the MOSFET sensor, andthe MOSFET sensor detects denaturated or renaturated double-strandednucleic acid at the melting point T_(m) of the immobilized nucleic acid.

In the present invention, a MOSFET is used as a sensor for detecting acharge variation before and after separation of double-stranded nucleicacid.

It is preferable that the MOSFET is located at the sidewalls or convexcorners of the micro-fluid channel. Unlike recently disclosed FET-basedbiomolecular sensors manufactured on a plain, a biomolecular sensoraccording to the present invention is formed as a 3-dimensional (3D)MOSFET sensor by bulk micromachining and diffusion and is disposed atthe convex corners of the micro-fluid channel. The biomolecular sensorhaving this structure is located in the flow path of nucleic acid,thereby reducing the area occupied by a detection unit including thesensor as well as sharply shortening detection time. Thus, more sensorscan advantageously be mounted within a small space.

According to the present invention, the MOSFET used as a DNA sensor ischaracterized by including a thin gold (Au) layer in source and drainregions on which self-aligned monolayers of thiol-substituted nucleicacids are immobilized. In particular, the MOSFET sensor has two sourceand drain sensors, each coated with a thin oxide layer and in turn withan Au layer, thereby resulting in a MOS structure. Thiol groups having aselective affinity to the Au layer are attached to the ends of thenucleic acid molecule so that the thiol-substituted nucleic acidmolecules are adsorbed to the Au layer. Thiol-substituted nucleic acidmolecules are adsorbed to the surface of the Au layer as self-assembledmonolayers (SAMs). SAMs mean organic monolayers which are spontaneouslyarranged on the surface of a substrate in a regular pattern and formchemical bonds with the substrate. Thus, an additional manufacturingdevice is not required to form SAMs. Currently available biomolecularsensors fail to provide a limited binding site to biomolecules and havea weak binding force. However, selective adsorption, which is employedin the present invention, of thiol-substituted biomolecules to Au in theform of SAMs can eliminate the drawbacks of the existing biomolecularsensors. In addition, the direct adsorption of thiol-substitutedbiomolecules to the surface of the sensor provides excellent performanceof detecting charge variations before and after reaction.

Also, the present invention is characterized in that nucleic acidsimmobilized on the MOSFET sensor include single-stranded DNA,single-stranded RNA, and single-stranded peptide nucleic acid (PNA).Single-stranded nucleic acids serve as probes that are hybridized to atarget nucleic acid injected through a sample loading unit. It ispreferable that discrete nucleic acid sequences are immobilized on eachMOSFET sensor.

In the present invention, the heater acts to raise the temperature ofthe thermal control and detection unit, and the thermal sensor acts tocontrol the operation of the heater. It is preferable that the heaterand the thermal sensor are located close to the MOSFET sensor foreffective control of the temperature which greatly affects nucleic acidsadsorbed to the MOSFET sensor.

To achieve the seventh object of the present invention, there isprovided a method for assaying nucleic acid mutation, the methodcomprising: immobilizing single-stranded nucleic acid probes on thesurface of a gate electrode of a MOSFET sensor; injecting a targetnucleic acid responsive to the immobilized single-stranded nucleic acidprobes into a sample loading unit and moving the target nucleic acid tothe MOSFET sensor along a micro-fluid channel; hybridizing the targetnucleic acid to the single-stranded nucleic acid probes immobilized onthe MOSFET sensor; gradually raising a temperature to separate thedouble-stranded nucleic acid which are hybridized, into two singlestrands; and measuring current-voltage characteristics of the gateelectrode of the MOSFET sensor.

In the nucleic acid mutation assay method according to the presentinvention, low-temperature nucleic acid hybridization is followed bygradual temperature rise to detect nucleic acid mutations. The thermalcontrol and detection unit disposed in the micro-fluid channel enablesreal-time nucleic acid mutation detection through accurate and precisetemperature adjustment.

Alternatively, the present invention provides a method for assayingnucleic acid mutation, the method comprising: immobilizingsingle-stranded nucleic acid probes on the surface of a gate electrodeof a MOSFET sensor; injecting a target nucleic acid responsive to theimmobilized single-stranded nucleic acid probes into a sample loadingunit and moving the target nucleic acid to the MOSFET sensor along amicro-fluid channel; keeping a temperature at which hybridization of thetarget nucleic acid to the single-stranded nucleic acid probes does notoccur; gradually dropping the temperature to renaturate single-strandednucleic acids into double-stranded nucleic acids which are hybridized;and measuring current-voltage characteristics of the gate electrode ofthe MOSFET sensor. In this case, high-temperature nucleic aciddenaturation is followed by gradual temperature drop to detect nucleicacid mutations.

The nucleic acid mutation assay device and method according to thepresent invention are effective in detecting nucleic acid mutations,particularly, single nucleotide polymorphism (SNP). Double-stranded DNAis denaturated into two single-strands at a temperature of 96° C. orgreater. The temperature of denaturation is relatively higher forhybrids having a perfectly matched base pair and relatively lower forhybrids having mismatched base pair. Based on this, a miniature systemof thermal control and detection in which reaction temperatures can beadjusted is manufactured by micro-electro mechanical system (MEMS)techniques. If discrete nucleic acid sequences are immobilized on eachDNA sensor implemented as a MOSFET sensor in the system according to thepresent invention, SNP of nucleic acid can be assessed by sensing thetemperature at which nucleic acid denaturation occurs, usingcorresponding temperature control units.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objects and advantages of the present invention will becomemore apparent by describing in detail preferred embodiments thereof withreference to the attached drawings in which:

FIG. 1 is a perspective view of a preferred embodiment of a moleculardetection device according to the present invention;

FIG. 2 is a partial top view of molecular detection chips according to apreferred embodiment of the present invention;

FIG. 3 is a partial perspective view of one molecular detection chip(metal oxide semiconductor field-effect transistor (MOSFET) sensor) ofFIG. 2;

FIG. 4 is a graph of the current-voltage (I-V) characteristics of themolecular detection chip of FIG. 2;

FIGS. 5 through 9 are perspective views illustrating each step of amethod for fabricating a molecular detection chip according to apreferred embodiment of the present invention;

FIG. 10 is a view illustrating immobilization of probes on a gateelectrode of the molecular detection chip;

FIG. 11 is a view of a preferred embodiment of a detection apparatus ofprobe DNAs immobilized using a spotting or photolithography techniqueaccording to the present invention;

FIG. 12 is a view of a preferred embodiment of a detection method of thehybridization of probe DNAs and a target DNA according to the presentinvention;

FIG. 13 is a diagram of a preferred embodiment of a nucleic acidmutation assay device according to the present invention in whichthermal control and detection units are disposed on the bottom of amicro-fluid channel;

FIG. 14 is a diagram of another preferred embodiment of the nucleic acidmutation assay device according to the present invention, in whichMOSFETs of a thermal control and detection unit, on which nucleic acidsare immobilized, are formed on the sidewalls or convex corners of amicro-fluid channel, and a thermal control and detection unit having aheater and a thermal sensor is disposed above the micro-fluid channel;

FIG. 15 shows fabrication of a MOSFET used in the thermal control anddetection unit of the nucleic acid mutation assay device according tothe present invention;

FIG. 16 is a graph of the current variation with respect to the amountof probe DNAs immobilized on a biochip according to the presentinvention; and

FIG. 17 is a graph of the current variation as a probe DNA and targetDNA are hybridized together according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A molecular detection chip, a molecular detection device employing themolecular detection chip, a method for fabricating the moleculardetection chip, and a molecular detection method using the moleculardetection device according to the present invention now will bedescribed more fully with reference to the accompanying drawings. Thisinvention may, however, be embodied in many different forms and shouldnot be constructed as being limited to the embodiments set forth herein;rather, these embodiments are provided as that this disclosure will bethorough and complete, and will fully convey the concept of theinvention to whose skilled in the art. In the drawings, the thickness oflayers are exaggerated for clarity, and like reference numerals are usedto refer to like elements throughout.

FIG. 1 is a perspective view of a preferred embodiment of the moleculardetection device according to the present invention. Referring to FIG.1, the molecular detection device includes a molecular detection kit 10and a molecular detection chip (not shown) mounted on the moleculardetection kit 10. The molecular detection kit 10 includes at least onesample loading unit 20 formed in the surface of a substrate 15, which isconnected to a chip mount region 40 through a fluid channel 30. Thewidth and depth of the fluid channel 30 are determined to be largeenough to allow a sample to flow by capillary action. In FIG. 1,reference numeral 50 denotes a sample dispenser, and reference numeral55 denotes a molecular sample.

Although not illustrated in FIG. 1, the molecular detection kit 10includes a variety of electric devices that can communicate with themolecular detection chip mounted on the chip mount region 40 throughelectric signals.

In the embodiment of FIG. 1, the molecular detection chip is describedas being detached from the molecular detection kit 10, but it may alsobe attached to the same for measurement. Alternatively, the moleculardetection chip may be built into the molecular detection kit 10 ifnecessary.

FIG. 2 is a partial top view of molecular detection chips according to apreferred embodiment of the present invention to be mounted in the chipmount region 40 of the molecular detection kit 10 of FIG. 1. Referringto FIG. 2, each molecular detection chip includes a micro-fluid channel30′ connected to one end of the fluid channel 30 having the other endconnected to a corresponding sample loading unit 20 of the moleculardetection kit 10 of FIG. 1, and a sample exhaust unit 60 connected tothe opposite end of the micro-fluid channel 30′. Source electrodes 150S,drain electrodes 150D, and gate electrodes 150G are formed on thesurface of the molecular detection chip, and interconnects 150 areconnected to the source electrodes 150S or drain electrodes 150D.

The structure of the molecular detection chip according to the preferredembodiment of the present invention shown in FIG. 2 will be described ingreater detail with reference to FIG. 3, which is a partial perspectiveview of one molecular detection chip of FIG. 2.

Referring to FIG. 3, a micro-fluid channel 105 as a sample flow path isformed on the surface of a semiconductor substrate 100. Four metal oxidesilicon field-effect transistors (MOSFETs) are formed on the sidewallsof the micro-fluid channel 105. The molecular detection chip accordingto the present invention can include at least one MOSFET. Each of theMOSFETs includes source and drain regions 120S and 120D formed on thesidewalls of the micro-fluid channel 105 by doping impurity ions, and achannel region 130 defined on the convex corners of the micro-fluidchannel 105 by the source and drain regions 120S and 120D. A gateelectrode 150G is formed in each channel region 130 with a gateinsulating layer 132 interposed therebetween. The gate electrode 150G ispreferably formed of a thin gold (Au) layer such that probes, to which atarget molecule is coupled, are stably immobilized as self-assembledmonolayers. Interconnects 140 for connecting the source and drainregions 120S and 120D to source and drain electrodes 150S and 150D,respectively, are formed on the surface of the substrate 100.

A current-voltage (I-V) characteristic curve for the molecular detectionchip of FIG. 3 is shown in FIG. 4. As shown in FIG. 4, the preferredembodiment of the molecular detection chip according to the presentinvention shows normal electrical characteristics for a MOSFET.

A method for fabricating the molecular detection chip of FIG. 3 will bedescribed with reference to FIGS. 5 through 9. Referring to FIG. 5, asilicon oxide layer 102 is formed on a semiconductor substrate 100 to athickness of 1.5-2.0 μm. It is preferable that the semiconductorsubstrate 100 is an n-type silicon substrate with a (100) directionplane.

Next, as shown in FIG. 6, a micro-fluid channel 105 having at least oneconvex corner is formed in the semiconductor substrate 100 byphotolithography. The reason for the need of at least one convex cornerwill be described in a subsequent process. In the present embodiment ofFIG. 6, although the micro-fluid channel 105 is formed to be longer inthe direction of line a-a′ and relatively shorter, but long enough toform at least one convex corner, in the direction of line b-b′, it willbe appreciated that the micro-fluid channel 105 may be stretched in thedirection of line b-b′ to form bidirectional, crossed micro-fluidchannels if needed.

Next, as shown in FIG. 7A, the entire surface of the substrate 100 isdoped with impurity ions. P-type impurity ions are used for the dopingof the substrate 100. For example, the substrate 100 may be doped withboron at a concentration of 10¹⁶-10¹⁸atoms/cm². Based on oxide removalrate with respect to the thickness of a natural silicon oxide layer, bycontrolling implantation conditions, such as temperature and time, onlythe sidewalls of the micro-fluid channel 105, not the bottom, can bedoped with impurity ions. Through a subsequent diffusion process, animpurity diffusion region 110 is formed only on the sidewalls of themicro-fluid channel 105, as shown in FIG. 7B, which is a partialenlarged view of FIG. 7A.

The resultant structure of FIG. 7B is etched using an etching solution.A trimethylammonium hydroxide (TMAH) solution is preferably used as theetching solution. Since only the convex corners of the micro-fluidchannel 105 are selectively etched, the impurity diffusion region 110 ispartially opened, resulting in an open region serving as a channelregion 130 at the corners of the micro-fluid channel 105, as shown inFIG. 8A. As a result, a source region 120S, a drain region 120D, and thechannel region 130 are formed.

The reason for selective etching on the convex corners of themicro-fluid channel 105 lies in the fact that crystal orientationdirection differs from one region to another region that is made contactwith the etching solution, as shown in FIG. 8B. The bottom surface ofthe micro-fluid channel 105 is formed by the (100) direction plane, likethe original silicon substrate 100, whereas the etched sidewalls of themicro-fluid channel 105 are comprised of a (111) direction plane.Therefore, the convex corners of the micro-fluid channels 105, at whichthe (111) direction planes meet, are selectively etched at apredetermined angle.

Whether the impurity diffusion region 110 is opened to form the channelregion 130 can be easily detected by etching the substrate 100 with anapplication of reverse bias voltage. When the impurity diffusion region110 is shorted, current does not flow although a reverse bias voltage isapplied to the impurity diffusion region 110. However, current starts toflow as soon as the impurity diffusion region 110 is opened at thecorners of the micro-fluid channel 105 by etching. An etch stop point iseasily determined based on current variations.

Lastly, a gate insulation layer 132 is formed on the substrate 100 inwhich the source region 120S, the drain region 120D, and the channelregion 130 are formed, as shown in FIG. 9, and a gate electrode 150G isformed. The gate insulation layer 132 is formed of a silicon oxide layerto have a thickness of 300-800 Å. The gate electrode 150G is formed as athin gold (Au) layer to enable easy immobilization of high-densityprobes thereon. It is preferable that a thin chromium (Cr) layer isformed prior to the formation of the thin Au layer to improve adhesionof the gate electrode 150G made of Au to the gate insulation layer 132.Interconnects 140 connected to the source regions 120S or drain regions120D, and source and drain electrodes 150S and 150D are formed throughgeneral processes, thereby forming a molecular detection chip.

A molecular detection method using the molecular detection deviceaccording to the present invention now will be described with referenceto FIGS. 1 and 3. Initially, the surface of the molecular detection chipis cleaned with a piranha solution (a mixture of H₂SO₄ and S₂O in aratio of 3:1) and then with deionized water, followed by drying usingnitrogen gas and treating with UV ozone. As a result, organic substanceis removed from the surface of the chip. The surface of the moleculardetection kit 10 is cleaned in the same manner as the surface of thechip. After the cleaning step is finished, the molecular detection chipof FIG. 3 is mounted on the chip mount region 40 of the moleculardetection kit 10 of FIG. 1. Next, a probe sample is applied into thesample loading unit 20 of FIG. 1. A probe sample having a thiol group atits end is used. The probe sample moves along the fluid channels 30 ofFIG. 1 connected to the sample loading unit 20 by capillary action. Theprobe sample is immobilized on the top of the gate electrode 150G ofeach MOSFET formed on the sidewalls of the micro-fluid channel 105 ofFIG. 3 in the form of self-assembled monolayers while passing along themicro-fluid channel 105 connected to each of the fluid channels 30.Sample probes being immobilized on the surface of the gate electrode 150are shown in FIG. 10. When probes are immobilized as self-assembledmonolayers on the top of the gate electrode 150G formed of Au, thiolgroups serving as linkers are used. In FIG. 10, reference numeral 210denotes thiol groups, and reference numeral 220 denotes probes. Suchselective immobilization of self-assembled monolayers of probes on thegate electrode 150G made of Au using the thiol groups 210 as linkersresults in a highly dense and well-aligned structure of the immobilizedprobes with strong binding force, as shown in FIG. 10. As a result,stable coupling with a target molecule can be maintained through asubsequent reaction process.

Suitable probes may include nucleic acids, proteins, enzyme substrates,adjuvants, and oligosaccharides, which are responsive to a targetmolecule. Single-stranded deoxyribonucleic acid (DNA), single-strandedribonucleic acid (RNA), or single-stranded peptide nucleic acid (PNA)can be used as a nucleic acid. Suitable proteins include agonists tocell membrane receptors, antagonists to cell membrane receptors, toxins,virus epitopes, hormones, peptides, enzymes, and monoclonal antibodies.

After the probe immobilization on the gate electrode 150G, voltage andcurrent flowing across the gate electrode 150G are measured.

Next, a target sample is applied into the sample loading unit 20 ofFIG. 1. The target sample refers to biomolecular samples from liveorganisms and synthetic samples. The target sample moves along the fluidchannel 30 connected to the sampling loading unit 20 and finally passesthrough the micro-fluid channel 105 of FIG. 3 of the molecular detectionchip connected to the fluid channel 30. The target sample reacts withthe probes 220 of FIG. 10 while passing through the micro-fluid channel105.

Current-Voltage (I-V) characteristics on the gate electrode 150G aremeasured, and whether or not the target sample is coupled to the probesis determined by measuring the change in I-V characteristics before andafter the sample-to-probe binding.

For another sample detection, the probes used are removed from the gateelectrode 150G made of Au. The probes used can be removed by washing thegate electrode 150G using the piranha solution (a 3:1 mixture of H₂SO₄and S₂O).

Next, the same processes described above are repeated for the new targetsample using probes that are responsive to the target sample to detectthe target sample.

According to the present invention, the immobilization of probes on thegate electrode 150G as self-assembled monolayers is easy to perform withexcellent reproducibility. Separation of the probes for another sampledetection is also easy. Thus, the molecular detection device accordingto the present invention is advantageous in that a variety of probes caneasily be used.

According to the present invention, a MOSFET sensor can be formed on thesidewalls of a micro-fluid channel, as shown in FIG. 3, or can beunderneath the surface of a general planar biochip. FIG. 11 shows adetection system for measuring the quantity of DNAs immobilized on a DNAchip using the MOSFET sensor installed underneath the DNA chip surface.In FIG. 11, (A) shows the DNA chip manufactured using a spotting orphotolithography technique, (B) shows an enlarged region of one spot,and (C) shows a structure of the MOSFET sensor including a source region250S, a drain region 250D, and a gold (Au)-film deposited gate region250G.

FIG. 12 shows a detection system for quantifying the immobilization ofprove DNAs and target DNAs in each spot region. In FIG. 12, (A) showsonly single-stranded probe DNAs immobilized on the Au region of a DNAchip (i.e., the surface of a MOSFET sensor), and (B) shows the probeDNAs being hybridized to the target DNAs.

In manufacturing a spotting chip or photolithography chip, glass isgenerally used for a substrate. In the present invention, a siliconwafer is preferred for the substrate. Silicon wafers have electricalcharacteristics as well as excellent mechanical properties, compared toglass, and thus can be applied for DNA immobilization and detectionsystems with excellent characteristics. By attaching a thiol-substitutedgroup to the end of probe DNAs, the probe DNAs can be selectivelyimmobilized only on the Au region patterned in a silicon wafer.

FIG. 13 shows a preferred embodiment of a nucleic acid mutation assaydevice according to the present invention, in which a plurality ofthermal control and detection units are disposed on the bottom of amicro-fluid channel, wherein each thermal control detection unitincludes a MOSFET sensor 300, a heater 400, and a thermal sensor 500. InFIG. 13, the arrows indicate the direction in which fluid flows, andstrands extending from the MOSFET sensor 300 are DNA strands. Inparticular, (a) indicates perfectly matched DNA hybrids, and (b), (c),and (d) indicate mismatched DNA hybrids.

FIG. 14 shows another preferred embodiment of the nucleic acid mutationassay device according to the present invention, in which MOSFET sensorsof the thermal control and detection unit, each having an electrode 300Eand a Au gate 300G, are disposed on the sidewalls or convex corners ofthe micro-fluid channel, and a micro-heater 400′ and micro-thermalsensor 500′ are mounted above the micro-fluid channel.

The present invention will be described in greater detail with referenceto the following embodiments. The following embodiments are forillustrative purposes and are not intended to limit the scope of theinvention.

Embodiment 1 Fabrication of Molecular Detection Device or Nucleic AcidMutation Assay Device

1. Formation of Micro-fluid Channel

In general, the body of the molecular detection device or the nucleicacid mutation assay device according to the present invention can beassembled using a method and material compatible with microfabricationtechniques. For example, the body of the molecular detection device orthe nucleic acid mutation assay device may include a polymer-based partformed by injection molding using a variety of polymers, or a pluralityof planar crystalline substrates formed of silicon, glass, etc. Avariety of wells or channels may be formed in a crystalline substratemade of, for example, silica, glass or silicon, by etching, milling,drilling, etc. These materials and methods are compatible withmicrofabrication techniques that are widely used in semiconductorrelated industries. Available microfabrication techniques include, forexample, electrodeposition, low-pressure vapor deposition,photolithography, etching, laser drilling, etc.

Photolithography is more compatible to etch substrates in themicrofabrication of the nucleic acid mutation assay device according tothe present invention. For example, a substrate is overlaid with aphotoresist and exposed through a photolithographic mask toelectromagnetic rays to form a photoresist pattern, which reflectschambers and/or channels to be formed in the molecular detection deviceor the assay device. The exposed substrate is etched to form desiredwells or channels. Next, the substrate on which the wells and/orchannels are formed is covered and bonded with another planar substrate.Suitable photoresists include polymethyl methacrylate (PMMA) and itsderivatives, electron beam resists such as poly(olefin sulfones) and thelike.

Preferably, the body of the molecular detection device or the nucleicacid mutation assay device may be formed by combining a part formed byinjection molding using, for example, plastic, and a planar silica orsilicon substrate that is etched. For example, a sample loading unit maybe formed by injection molding, whereas the micro-fluid channel or well,a thermal control and detection unit may be formed in a planar glass,silica, or silicon chip or substrate by etching in microfabrication.

2. Formation of DNA Detection Unit (Sensor)

FIG. 15 shows fabrication of a DNA detection unit (MOSFET sensor) to bemounted at convex corners of the micro-fluid channel. A nucleic acidinjected into a sample loading unit moves to a detection site of thesensor along the micro-fluid channel. First, an n-type silicon substrateis wet etched and followed by boron diffusion to form a p-type siliconregion on the etched sidewalls. The boron-doped region is divided intosource and drain regions by wet etching. To form a MOSFET structure, athin oxide layer is formed on the source and drain regions, and chromium(Cr) is deposited on the oxide layer for improved adhesion to Au. Next,Au is deposited on the Cr layer. Thiol-substituted nucleic acid probesare immobilized on the surface of the sensor. Thiol-substituted nucleicacids are selectively bound as self-assembled monolayers (SAMs) to onlythe Au layer on the surface of the sensor. The DNA detection unit forthe molecular detection device or the nucleic acid mutation assay deviceaccording to the present invention is fabricated by forming a thin Aulayer on a DNA sensor of MOSFET and then immobilizing thiol-substitutednucleic acids on the Au layer. SAMs of thiol-substituted nucleic acidattached to the Au layer are easily formed with excellentreproducibility. In addition, the variable opposite ends of the thiolgroups have wide applications. SAMs of the thiol-substituted nucleicacids are denser and are better assembled than other SAMs and are stablein a variety of reactions following the formation of SAMs due to astrong binding force.

3. Formation of Thermal Control Unit

A thermal control unit is formed by including a heater and a thermalsensor to control the temperature of a DNA detection unit (formed as aMOSFET sensor) which affects the structure of nucleic acid attached tothe same.

A thin-film resistive heater, which is known in the art, may be used asthe heater. The heater can be formed by depositing a metallic thin filmbelow, above, or inside the micro-fluid channel to be connected to apower source. As the thermal sensor, a bimetal thermocouple forgenerating temperature-dependent electromotive force (EMF), a resistivethermometer or thermistor including temperature-dependent electricresistance material, an IC thermal sensor, a quartz thermometer, etc.may be used.

Embodiment 2 Quantification of Probe DNA Immobilization

For quantification of the immobilization of probe DNAs, the chip of FIG.3 was mounted on the chip mount region 40 of the molecular detection kit10 of FIG. 1, and a voltage was applied. Synthetic DNA probes(5-thiol-GTTCTTCTCATCATC-3′) having substituted thiol groups at5′-terminal were loaded into the sample loading unit 20 in differentvolumes 20 μL, 40 μL, 80 μL, and 160 μL, respectively, and currentvariations over time were measured.

The results are shown in FIG. 16. As shown in FIG. 16, as the quantityof probe DNAs loaded into the sensor increases, the current droppinglevel flowing across the drain and source of the sensor proportionallyincreases. As a result, the current dropping level was 14 μA for 20 μLof the probe DNA (FIG. 16A), 17 μA for 40 μL of the probe DNA (FIG.16B), and 20 μA for 80 μL of the probe DNA (FIG. 16C). No increase incurrent dropping level was observed for more than 80 μL of the probe DNA(FIG. 16D).

Accordingly, by measuring the current level flowing across the gateelectrode of the sensor, probe DNAs immobilized on a DNA chip can bequantitatively measured. The quantification of the probe DNAs providesbasic data on reactions with a target DNA in the DNA chip.

Embodiment 3 Quantification of Probe DNA and Target DNA Hybridization

For quantification of the hybridization of probe DNAs to a target DNA, avoltage was applied to the lab-on-a-chip of FIG. 3, and synthetic DNAprobes (5′-thiol-GTTCTTCTCATCATC-3′) having substituted thiol groups at5′-terminal and a target DNA having a complementary sequence to thesynthetic DNA probe were sequentially loaded. Then, current variationsover time were measured.

The chip of FIG. 3 was mounted on the chip mount region 40 of themolecular detection kit 10 of FIG. 1, and current flowing through thechip was measured with the application of a voltage. Next, a phosphatebuffer solution was injected into the sample loading unit 20 of themolecular detection kit 10. Current measurement continued as thephosphate buffer solution was injected. Immediately after the bufferinjection, the current level increased, as shown by reference A of FIG.17. Next, the current level stabilized for about 30 minutes. Then probesof 15 basepair (bp) synthetic DNA (5′-thiol-GTTCTTCTCATCATC-3′) havingsubstituted thiol groups at 5′-terminal were loaded into the sampleloading unit 20. After the probe DNA was loaded, the current leveldropped, as shown by reference B of FIG. 17. This indicates that theprobe DNA has been immobilized as self-assembled monolayers on thesurface of the gate electrode 150G made of Au of FIG. 3. Thus,immobilization of probe DNA can be detected in situ. Observation ofcurrent variations continued until a sufficient current drop wasdetected, i.e., for about 3 hours, and then deionized water was loadedinto the same loading unit 20 to get rid of free non-immobilized probeDNA and the phosphate buffer solution. Next, tris-EDTA buffer wassupplied through the sample loading unit 20 and left for about 1 hourwhile monitoring current level variations to provide optimalhybridization conditions for probe and target DNAs. After the currentlevel dropped to a level corresponding to reference C of FIG. 17, atarget DNA, which is complementary to the probe DNA, was loaded into thesample loading unit 20, followed by a current level measurement. Asshown in FIG. 17, after loading of the target DNA into the chip, a dropof current level of the chip occurred. Thus, hybridization betweentarget and probe DNAs can be detected in situ.

The molecular detection chip according to the present invention isdesigned to have MOSFETs on the sidewalls of a micro-fluid channel sothat it is possible to highly integrate the molecular detection chipwith increased detection sensitivity. In addition, immobilization ofprobes directly on the surface of a gate electrode in the form ofself-assembled monolayers ensures the molecular detection chip to checkfor the immobilization of probes and coupling of a target molecule tothe probes in situ. The immobilization of probes as self-assembledmonolayers provides a dense and stable coupling structure with highreproducibility. The immobilized probes can easily be separated foranother sample detection. The molecular detection device providesreliable detection results and can detect a variety of target moleculesby selectively using appropriate probes.

According to the present invention, the probes immobilized on thebiochip can be quantitatively measured using MOSFET sensors built in thebiochip. In addition, binding of the probes and a target sample can bequantitatively measured. For a DNA chip, immobilization of probe DNAsand hybridization to the target DNA can be accurately measured at thesame time using the MOSFET sensor. Therefore, DNA chips for commercialuses can be manufactured without an increase in the manufacturing cost.

In addition, according to the present invention, a heater, a thermalsensor, and a DNA sensor are all built in the micro-fluid channel sothat temperature-based nucleic acid denaturation can be detected in realtime. A variety of types of nucleic acid mutations, particularly singlenucleotide polymorphisms (SNPs), can be effectively detected.

A conventional method for assaying the sequence of mutated DNA needsseparate equipment and machine tools for temperature-based DNA helixseparation and detection. It is difficult to detect DNAs separatedthrough accurate and precise temperature control using the conventionalmethod. According to the present invention, thermal control anddetection units are disposed in the micro-fluid channel so that DNAmutations can be detected in real time through accurate and precisetemperature adjustment by merely injecting a DNA sample into themicro-fluid channel. The temperature distribution is more uniformthroughout the micro-fluid channel, thereby improving reliability inassay results.

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A method for fabricating a molecular detectionchip, the method comprising: forming an oxide layer on the surface of asemiconductor substrate; forming at least one micro-fluid channel havingsidewalls comprising at least one convex corner by etching the surfaceof the semiconductor substrate; forming an impurity diffusion region bydoping the sidewalls of the at least one micro-fluid channel withimpurity ions; defining a channel region of a metal oxide silicon-fieldeffect transistor (MOSFET) formed in the convex corner and furtherdefining a source region and a drain region spaced apart from each otherand formed in the sidewalls of the convex corner by etching a portion ofthe impurity diffusion region formed on the sidewalls of the at leastone micro-fluid channel using an etching solution; forming an additionaloxide layer on the channel region; and forming a gate electrode on thechannel region.
 2. The method of claim 1, wherein defining the channelregion is performed by selectively etching the impurity diffusion regionalong the at least one convex corner.
 3. The method of claim 1, wherein,in defining the channel region, an etch stop point is determined bymeasuring current level variations with application of reverse biasvoltage to the semiconductor substrate.
 4. The method of claim 1,wherein the gate electrode on the channel region is formed using gold(Au).
 5. A method of fabricating a molecular detection chip comprising:forming an oxide layer on a surface of a semiconductor substrate;forming at least one micro-fluid channel on the semiconductor substrate,wherein the at least one micro-fluid channel comprises sidewallscomprising at least one convex corner formed by etching the surface ofthe semiconductor substrate and the at least one micro-fluid channelserves as a flow path of a molecular sample on the semiconductorsubstrate; forming an impurity diffusion region by doping the sidewallsof the at least one micro-fluid channel with impurity ions; defining achannel region of a metal oxide silicon field-effect transistor(MOSFET), wherein the MOSFET comprises the channel region formed in theconvex corner, a source region and a drain region spaced apart from eachother and formed in the sidewalls of the convex corner, by etching aportion of the impurity diffusion region formed on the sidewalls of theat least one micro-fluid channel using an etching solution; forming anadditional oxide layer on the channel region; and forming a gateelectrode on the channel region.
 6. The method of claim 5, whereindefining the channel region is performed by selectively etching theimpurity diffusion region along the at least one convex corner.
 7. Themethod of claim 5, wherein, in defining the channel region, an etch stoppoint is determined by measuring current level variations withapplication of reverse bias voltage to the semiconductor substrate. 8.The method of claim 5, wherein the gate electrode on the channel regionis formed using gold (Au).
 9. A method of fabricating a moleculardetection device comprising: forming a molecular-sample loading unit onthe surface of a substrate; forming at least one micro-fluid channel onthe semiconductor substrate, wherein the at least one micro-fluidchannel comprises sidewalls comprising at least one convex corner formedby etching the surface of the semiconductor substrate and the at leastone micro-fluid channel serves as a flow path of a molecular sample onthe semiconductor substrate; and forming a metal oxide silicon-fieldeffect transistor (MOSFET) sensor connected to the other end of themicro-fluid channel and on which a molecular probe is immobilized,wherein the MOSFET comprises a channel region formed in the convexcorner, a source region and a drain region spaced apart from each otherand formed in the sidewalls of the convex corner and a gate electrodeformed on the channel region.
 10. A method of claim 1, whereinthio-substituted probes are immobilized as self-assembled monolayers onthe surface of the gate electrode.